This is the U.S. National Phase under 35 U.S.C. xc2xa7371 of International Patent Application PCT/BE99/00089, xe2x80x9cpublished in English under PCT Article 21(2)xe2x80x9d filed Jul. 9, 1999, which claims priority of European application EP 98870159.5, filed Jul. 10, 1998.
The present invention is related to a method of genetic modification of a wild type viral sequence, for reducing or suppressing deleterious properties of plants or plant cells transformed by said wild type viral sequence.
The present invention is also related to the modified viral sequence obtained by said method, and to the plant and the plant cell comprising said modified viral sequence.
The widespread viral disease of the sugar beet plant (Beta vulgaris) called Rhizomania is caused by a furovirus, the beet necrotic yellow vein virus (BNYVV) (1, 2) which is transmitted to the root of the beet by the soilborne fungus Polymyxa betae (3).
The disease affects significantly acreages of the area where the sugar beet plant is grown for industrial use in Europe, USA and Japan and is still in extension in several places in Western Europe (4, 5).
Since 1986, number of reports and publications have described the use of isolated viral nucleotidic sequences expressed in plants to confer a high level of tolerance against a specific infectious virus or even to confer a broad spectrum type of resistance against a number of related viruses (6, 7, 8). One of the most documented viral resistance strategies based on genetic engineering, in many cultivated species such as potato, squash, cucumber or tomato, is the use of the viral nucleotidic sequence which under the control of plant regulatory elements, encodes the coat-protein of the target virus (9).
However, in coat-protein mediated resistance, the expression of a certain level of resistance in the transgenic plant might be attributed to different mechanisms such as RNA co-suppression and not necessarily to the production of the protein sequence.
In general, the virus sequence will be transformed in an appropriate cell or tissue culture of the plant species using an Agrobacterium mediated transformation system or a direct gene transfer method according to the constraints of the tissue culture or cell culture method which can be successfully applied in a given species. A whole plant will be regenerated and the expression of the transgene will be characterised.
Though sugar beet is known as a recalcitrant species in cell culture, limiting the extent of practical genetic engineering applications in that species, there are number of isolated reports of successful transformation and regeneration of whole plants (38). A few examples of engineering tolerance to the BNYVV by transforming and expressing the BNYVV coat-protein sequence in the sugar beet genome have also been published (11, WO91/13159) though they rarely report data on whole functional transgenic sugar beet plants (12). In particular, reports show limited data on the level of resistance observed in infected conditions with transgenic sugar beet plants transformed with a gene encoding a BNYVV coat-protein sequence (13, 14).
A complete technology package including a sugar beet transformation method and the use of the expression of the BNYVV coat-protein sequence as a resistance source in the transgenic sugar beet plant obtained by said transformation method has been described in the Patent Application WO91/13159.
Based on the information published, it can not be concluded that the coat-protein mediated resistance mechanism provides any potential for conferring to the sugar beet plant a total immunity to the BNYVV-infection by inhibiting the virus multiplication and diffusion mechanisms completely. To identify a resistance mechanism which significantly blocks the spread of the virus at the early stage of the infection process would be a major criteria of success to develop such a transgenic resistance. In addition, such resistance would diversify the mechanisms of resistance available.
Because the disease is shown to expand in many countries or areas, at a speed depending upon the combination of numerous local environmental and agricultural factors, there is a major interest to diversification and improvement of the genetic resistance mechanisms which may, alone or in combination, confer a stable and long lasting resistance strategy in the current and future varieties of sugar beet plants which are grown for industrial use.
The genome of beet necrotic yellow vein furovirus (BNYVV) consists of five plus-sense RNAs, two of which (RNAs 1 and 2) encode functions essential for infection of all plants while the other three (RNAs 3, 4 and 5) are implicated in vector-mediated infection of sugar beet (Beta vulgaris) roots. Cell-to-cell movement of BNYVV is governed by a set of three successive, slightly overlapping viral genes on RNA 2 known as the triple gene block (TGB), which encode, in order, the viral proteins P42, P13 and P15 (gene products are designated by their calculated Mr in kilodalton).
In the following description, the TGB genes and the corresponding proteins will be identified by the following terms: TGB-1, TGB-2, TGB-3 or by their encoded viral protein number P42, P13 and P15. TGB counterparts are present in other furoviruses and in potex-, carla- and hordeiviruses (15, 18, 19, 20, 21 and 22). In the enclosed table 1 are represented viruses having a TGB-3 sequence, the molecular weight of TGB-3 of said viruses, their host and references.
It has been shown previously that independent expression of P15 from a viral-RNA replication species known as a xe2x80x9crepliconxe2x80x9d, derived from BNYVV RNA 3, inhibits infection with BNYVV by interfering cell-to-cell movement (16).
In order to introduce a virus comprising a TGB-3 nucleic acid sequence into a plant cell or a plant, it has been proposed to incorporate a nucleic acid construct comprising said TGB-3 nucleic acid sequence operably linked to one or more regulatory sequences active in said plant (WO98/07875).
However, while expression of wild type TGB-3 viral sequence in a transgenic plant allows the blocking of said viral infection, the presence of said wild type sequence may induce deleterious effects on the agronomic properties of transformed plants or plant cells.
The present invention aims to provide a new method for inducing a genetic modification of a wild type viral sequence involved in the multiplication and diffusion mechanisms of virus infecting plants, in order to reduce or suppress the possible deleterious effects upon plants or plant cells transformed by said viral sequence.
Another aim of the present invention is to provide a method to obtain such a modified viral sequence which blocks virus infection when it is incorporated into a plant or a plant cell.